Significant gaps in immunity to polio, measles, and rubella may exist in adults in Cambodia and threaten vaccine-preventable disease (VPD) elimination and control goals, despite high childhood vaccination coverage. We conducted a nationwide serological survey during November–December 2012 of 2154 women aged 15–39 years to assess immunity to polio, measles, and rubella and to estimate congenital rubella syndrome (CRS) incidence. Measles and rubella antibodies were detected by IgG ELISA and polio antibodies by microneutralization testing. Age-structured catalytic models were fitted to rubella serological data to predict CRS cases. Overall, 29·8% of women lacked immunity to at least one poliovirus (PV); seroprevalence to PV1, PV2 and PV3 was 85·9%, 93·4% and 83·3%, respectively. Rubella and measles antibody seroprevalence was 73·3% and 95·9%, respectively. In the 15–19 years age group, 48·2% [95% confidence interval (CI) 42·4–54·1] were susceptible to either PV1 or PV3, and 40·3% (95% CI 33·0–47·5) to rubella virus. Based on rubella antibody seroprevalence, we estimate that >600 infants are born with CRS in Cambodia annually. Significant numbers of Cambodian women are still susceptible to polio and rubella, especially those aged 15–19 years, emphasizing the need to include adults in VPD surveillance and a potential role for vaccination strategies targeted at adults.